Activation of mGluR5 Induces Rapid and Long-Lasting
Protein Kinase D Phosphorylation in Hippocampal
Neurons
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Krueger, Dilja D., Emily K. Osterweil, and Mark F. Bear.
“Activation of mGluR5 Induces Rapid and Long-Lasting Protein
Kinase D Phosphorylation in Hippocampal Neurons.” Journal of
Molecular Neuroscience 42.1 (2010): 1–8.
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http://dx.doi.org/10.1007/s12031-010-9338-9
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Author Manuscript
J Mol Neurosci. Author manuscript; available in PMC 2011 September 1.
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Published in final edited form as:
J Mol Neurosci. 2010 September ; 42(1): 1–8. doi:10.1007/s12031-010-9338-9.
Activation of mGluR5 Induces Rapid and Long-Lasting Protein
Kinase D Phosphorylation in Hippocampal Neurons
Dilja D. Krueger, Emily K. Osterweil, and Mark F. Bear
Howard Hughes Medical Institute, Picower Institute for Learning and Memory, Department of
Brain and Cognitive Sciences, Massachusetts Institute of Technology, 43 Vassar St, 46-3301,
Cambridge, MA 02139, USA
Abstract
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Metabotropic glutamate receptors (mGluRs), including mGluR5, play a central role in regulating
the strength and plasticity of synaptic connections in the brain. However, the signaling pathways
that connect mGluRs to their downstream effectors are not yet fully understood. Here, we report
that stimulation of mGluR5 in hippocampal cultures and slices results in phosphorylation of
protein kinase D (PKD) at the autophosphorylation site Ser-916. This phosphorylation event
occurs within 30 s of stimulation, persists for at least 24 h, and is dependent on activation of
phospholipase C and protein kinase C. Our data suggest that activation of PKD may represent a
novel signaling pathway linking mGluR5 to its downstream targets. These findings have important
implications for the study of the molecular mechanisms underlying mGluR-dependent synaptic
plasticity.
Keywords
mGluR5; Protein kinase D; DHPG; MPEP; Hippocampus; Primary culture
Introduction
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Metabotropic glutamate receptors (mGluRs) are widely expressed throughout the central
nervous system (Conn 2003), where they play an important role in synaptic plasticity. Group
I mGluRs, comprising mGluR1 and mGluR5, have been implicated in several forms of
synaptic plasticity, including long-term depression of synaptic transmission (Oliet et al.
1997; Huber et al. 2000) as well as priming of long-term potentiation (Cohen and Abraham
1996). The signaling pathways linking mGluRs to the molecular effectors of synaptic
plasticity are currently under active investigation. Group I mGluRs are Gq-coupled receptors
that can signal both through the canonical Gαq-phospholipase C (PLC) pathway and through
more recently identified G protein-independent mechanisms (Gerber et al. 2007). Signaling
pathways that are reported to be activated by group I mGluR stimulation and that have been
implicated in mGluR-dependent plasticity include ERK1/2, p38 MAPK, and the PI3K/Akt
pathway, although the relative contributions of each of these pathways remain to be
determined.
Protein kinase D (PKD) is a serine/threonine kinase that was originally described as an
atypical isoform of protein kinase C (PKC), PKCμ (Johannes et al. 1994), but is now
considered to belong to the Ca2+/calmodulin-dependent kinase superfamily based on
© Springer Science+Business Media, LLC 2010
mbear@mit.edu .
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substrate specificity and sequence homology in the catalytic domain (Valverde et al. 1994;
Rozengurt et al. 2005). PKD is expressed ubiquitously, and it has been implicated in
functions as diverse as regulation of transport from the trans-Golgi network to the plasma
membrane, cell motility and adhesion, cell proliferation and apoptosis, nuclear export of
histone deacetylases, and oxidative stress (for reviews, see Rozengurt et al. 2005; Avkiran et
al. 2008). In neurons, PKD in the Golgi was recently reported to play a role in dendritic
trafficking and the establishment of neuronal polarity and dendritic complexity (Yin et al.
2008; Bisbal et al. 2008; Czondor et al. 2009). PKD is activated by binding of diacylglycerol
(DAG) and phosphorylation by PKC at Ser-744 and Ser-748 (Iglesias et al. 1998), followed
by autophosphorylation at Ser-916, which is believed to correlate with PKD catalytic
activity (Matthews et al. 1999). A number of extracellular signals have been shown to
induce PKD phosphorylation and activation, including bombesin, bradykinin, vasopressin,
platelet-derived growth factor, and insulin-like growth factor (Rozengurt et al. 2005),
underlining the importance of this pathway in a variety of cellular functions.
Here, we demonstrate that activation of mGluR5 using the group I mGluR-specific agonist
3,5-dihydroxyphenylglycine (DHPG) results in phosphorylation of PKD at Ser-916 in
hippocampal cultures and slices. To our knowledge, this represents the first report of a link
between glutamate receptors and PKD, and it raises the possibility that PKD may be one of
the pathways important in mGluR-dependent plasticity.
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Materials and Methods
Animals
Pregnant E18 Sprague-Dawley rats were obtained from Charles River Laboratories. C57BL/
6J mice were bred at MIT. All animals were treated in accordance with NIH and MIT
guidelines.
Pharmacological Activators and Inhibitors
The following drugs were used in this study (all from Tocris Biosciences unless stated
otherwise): APV, BAPTA-AM, CNQX, (RS)-3,5-dihydroxyphenylglycine (DHPG),
genistein, GF109203X, Go6976, LY367385, 2-methyl-6-(phenylethynyl)pyridine
hydrochloride (MPEP), PP2, rapamycin, SB203580, tetrodotoxin (TTX), U0126, U73122,
wortmannin, and xestospongin C (Sigma-Aldrich).
Primary Hippocampal Cultures
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Primary hippocampal cultures were prepared from E18 rat embryos as previously described
(Krueger and Nairn 2007). Briefly, hippocampi were isolated in ice-cold dissociation
medium and digested for 1 h in 0.01% papain (Worthington). The tissue was triturated, and
cells were collected by centrifugation and resuspended in plating medium (neurobasal
medium containing B27 supplement, GlutaMax, 1% penicillin/streptomycin, and 10% fetal
bovine serum, all from Invitrogen). Cells were plated at high density (250,000 cells per 3.8
cm2 well) onto tissue culture plates coated with poly-L-lysine. On the following day, the
medium was replaced with neurobasal medium containing all of the above supplements
except fetal bovine serum, and cells were maintained in this medium at 37°C, 5% CO2 for
21 days until use to allow synapse maturation.
Cell Culture Stimulation and Harvest
For stimulation, DHPG (or vehicle control) was added to a final concentration of 100 μM
(unless otherwise indicated) and incubated at 37°C, 5% CO2 for the indicated period of time.
For experiments involving time points greater than 5 min after stimulation, the medium
containing DHPG was removed after 5 min and replaced with medium without DHPG, and
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cells were again incubated at 37°C, 5% CO2 for the indicated period of time from the onset
of stimulation. For experiments involving pre-incubation with inhibitors, the inhibitor (or
vehicle control) was added 30 min prior to stimulation, and it remained present throughout
the duration of the stimulation. To harvest cells for immunoblotting, plates were washed
with ice-cold phosphate buffered saline, and cells were lysed by addition of 50-μl lysis
buffer per well (1% SDS, 50 mM Tris, 1 mM EDTA, 1 mM EGTA, pH 7.4, containing
freshly added protease and phosphatase inhibitor cocktails (EMD Chemicals) at a dilution of
1:100).
Slice Preparation and Stimulation
Hippocampal slices were prepared from male P26–P30 mice as previously described (Dolen
et al. 2007). Briefly, hippocampi were rapidly dissected into ice-cold artificial cerebral
spinal fluid (ACSF; in millimolar: NaCl, 124; KCl, 3; NaH2PO4, 1.25; NaHCO3, 26;
dextrose, 10; MgCl2, 1.0; CaCl2, 2, saturated with 95% O2 and 5% CO2). Acute
hippocampal slices (500 μm) were prepared using a Stoelting Tissue Slicer and incubated
for 4 h in 32.5°C ACSF. For stimulation, slices were incubated in 100 μM DHPG in ACSF
for 5 min. They were then either homogenized in Laemmli sample buffer (for analysis of
whole tissue homogenate) or processed further to obtain synaptoneurosomes as described
below.
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Synaptoneurosomes—Synaptoneurosomes were isolated from slices as described (Chen
and Bear 2007). Briefly, slices were homogenized, passed through 2×105-μm meshes, then
1×5-μm mesh, and the resulting samples were spun at 1,000×g for 10 min at 4°C. The
resulting pellets were then washed, spun at 1,000×g, and resuspended in Laemmli sample
buffer containing protease and phosphatase inhibitors.
Immunoblotting
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Lysates were processed for immunoblotting as previously described (Krueger and Nairn
2007). Briefly, samples were boiled in Laemmli sample buffer, resolved on 10% SDSPAGE gels, transferred onto nitrocellulose membranes, and stained for total protein using a
Memcode assay (Pierce, Rockford, IL, USA). Membranes were blocked in 5% fat-free milk
and then incubated in primary antibody (phospho-PKD Ser-916 or total PKD, both from
Cell Signaling Technology), followed by secondary antibody (goat anti-rabbit-IRDye680,
Rockland Immunochemicals). Blots were scanned on an Odyssey Infrared Imager (LiCor
Biosciences), and the signal intensity for each sample was quantified using the Odyssey 2.0
software. Each sample value was divided by the total protein loading value for the
corresponding lane and then normalized to the average sample value of all lanes on the same
blot to correct for blot-to-blot variance.
Statistical Analysis
Group comparisons for experiments involving time courses were performed using repeated
measures analysis of variance (ANOVA) for time and stimulation. Group comparisons for
the DHPG dose–response curve were performed using one-way ANOVA for stimulation.
Group comparisons for experiments involving inhibitors were performed using two-way
ANOVA for stimulation and inhibitor. Post-hoc analysis was performed using two-tailed
Student's t tests. Group comparisons for the slice and synaptoneurosome experiments were
performed using two-tailed paired Student's t tests. All data are expressed as mean ± SEM.
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Results and Discussion
Stimulation of Hippocampal Cultures with DHPG Induces Phosphorylation of PKD
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To determine whether activation of group I mGluRs can result in phosphorylation of PKD,
primary hippocampal cultures were stimulated with the group I mGluR agonist DHPG, and
they were subsequently assessed for PKD phosphorylation by immunoblotting. Since the
intention of this study was to investigate signaling pathways that may contribute to mGluRmediated plasticity, a stimulation paradigm was chosen that was previously shown to induce
long-term depression in hippocampal slices (Huber et al. 2001). Specifically, cultures were
incubated with 100 μM DHPG for 5 min, and the DHPG-containing medium was then
removed and replaced with DHPG-free medium, followed by a further incubation period
that lasted for up to 60 min from the onset of stimulation. This stimulation paradigm resulted
in a robust increase in phosphorylation of PKD at the autophosphorylation site Ser-916 (Fig.
1a), which is thought to correlate with PKD catalytic activity (Matthews et al. 1999). A
doublet band was observed by immunoblot, corresponding to phosphorylation of the two
isoforms PKD1 and PKD2. Since the two bands were affected in a similar manner by DHPG
stimulation, they were quantified together to yield an overall measure of PKD
phosphorylation. The DHPG-induced PKD phosphorylation was greatest at 5 min, but lasted
for at least 60 min after the onset of stimulation (Fig. 1a, b; repeated measures ANOVA for
treatment, p<0.0001; treatment×time interaction, p<0.05). Interestingly, total levels of PKD1
showed a slight but significant decrease at 5 min after DHPG stimulation (Fig. 1a, c;
repeated measures ANOVA for treatment, p<0.05; treatment×time interaction, not
significant). Since the magnitude of the decrease was negligible compared to the increase in
PKD phosphorylation, it was not further considered in this study.
DHPG-Induced PKD Phosphorylation Is Rapid and Sustained
Since PKD phosphorylation was already elevated to 1,000% of baseline after 5 min of
DHPG stimulation, the first time point previously measured, a shorter time course was
conducted to further investigate the onset of phosphorylation. To this end, cultures were
stimulated for 30 s, 1, 2.5, and 5 min with 100 μM DHPG. Even after 30 s, PKD
phosphorylation levels were increased almost 400%, and they continued to rise across the
time course assessed (Fig. 1d; repeated measures ANOVA for treatment, p< 0.0001;
treatment×time interaction, p<0.0001). This suggests that the onset of PKD phosphorylation,
and hence group I mGluR activation, occurs extremely rapidly in response to DHPG
stimulation.
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Since PKD phosphorylation was still elevated 60 min after a brief DHPG application, a
more extended time course was conducted to investigate how long this phosphorylation
would last. Cultures were stimulated with 100 μM DHPG for 5 min, followed by a DHPGfree period lasting for up to 24 h. As observed previously, PKD phosphorylation was
robustly increased to ~750% of baseline 1 h after stimulation. By 3 h after stimulation,
phosphorylation levels were reduced to ~250% of baseline, and they were sustained at this
level for up to 24 h (Fig. 1e; repeated measures ANOVA for treatment, p<0.0001;
treatment×time interaction, p<0.0001). These results imply that either group I mGluRs
remain active for 24 h after stimulation, possibly due to low levels of DHPG remaining
bound to the receptors, or that PKD phosphorylation is maintained independently of mGluR
activation.
PKD Phosphorylation Is Highly Sensitive Even to Low Concentrations of DHPG
The DHPG concentration applied in the initial experiments, 100 μM, was chosen based on
previous studies using DHPG to induce mGluR-dependent synaptic plasticity (Huber et al.
2001). However, 50 and 10 μM are also commonly reported to induce long-term depression
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and priming of long-term potentiation, respectively (Huber et al. 2001; Mellentin et al.
2007). To establish a dose–response curve for DHPG-mediated PKD phosphorylation,
cultures were stimulated with control, 1, 10, 50, or 100 μM DHPG for 5 min. Even at 1 μM
DHPG, a trend toward an increase in PKD phosphorylation was observed, and 10 μM
DHPG resulted in close to maximal phosphorylation (Fig. 1f; oneway ANOVA for
stimulation, p<0.0001; post-hoc analysis, control vs. 1 μM, p=0.081, control vs. 10, 50, and
50 μM, p<0.0001). These data indicate that PKD phosphorylation is highly sensitive even to
low levels of group I mGluR activation. For the sake of consistency, however, all further
experiments were performed at the same 100-μM DHPG dose used in the initial
characterization.
DHPG-Induced Phosphorylation of PKD Occurs Through mGluR5 but not mGluR1
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DHPG is a general agonist of group I mGluRs, including mGluR1 and mGluR5. In order to
identify which of these receptors mediates the observed PKD phosphorylation, cultures were
pre-incubated with either a specific antagonist of mGluR1 (LY367385, 50 μM) or of
mGluR5 (MPEP, 2 μM), and they were then stimulated with DHPG for 5 min. Preincubation with the mGluR5 antagonist MPEP completely blocked DHPG-induced PKD
phosphorylation (Fig. 2a and Table 1; two-way ANOVA for inhibitor, p< 0.0001;
inhibitor×stimulation interaction, p<0.0001; post-hoc analysis MPEP/control vs. MPEP/
DHPG, p=0.115). Conversely, pre-incubation with the mGluR1 antagonist LY367385 had
no effect of DHPG-induced PKD phosphorylation (Fig. 2b and Table 1; two-way ANOVA
for inhibitor, p=0.916; inhibitor×stimulation interaction, p= 0.474). These data suggest that
DHPG-induced stimulation of PKD phosphorylation occurs through mGluR5, not mGluR1.
DHPG-Induced Phosphorylation of PKD Is Dependent on Phospholipase C Activation
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One question arising from the above observations concerns the signaling pathways linking
mGluR5 stimulation to PKD phosphorylation. Group I mGluRs are G-protein coupled
receptors that can signal through a canonical Gαq-phospholipase Cβ (PLCβ) signaling
pathway, but also through more recently identified alternative pathways (Gerber et al. 2007).
To determine whether PLCβ is required for DHPG-induced PKD phosphorylation, cultures
were pre-incubated with a PLC inhibitor (U73122, 50 μM), followed by DHPG stimulation
for 5 min. Pre-incubation with the PLC inhibitor resulted in a significant reduction in
DHPG-induced PKD phosphorylation (Fig. 2c and Table 1; two-way ANOVA for inhibitor,
p<0.0001; inhibitor×stimulation interaction, p<0.0001), although a small DHPG-induced
increase was still detected in the presence of the inhibitor (post-hoc analysis U73122/control
vs. U73122/DHPG, p<0.0001). This may be due either to incomplete inhibition of PLCβ by
U73122, or to the activation of alternative signaling pathways that bypass the Gαq-PLCβ
pathway.
DHPG-Induced Phosphorylation of PKD Is Dependent on PKC but not Calcium Signaling
Activation of PLCβ results in production of the second messengers IP3 and DAG, which in
turn lead to release of calcium and activation of PKC. To test whether either calcium release
or PKC activation were required for DHPG-stimulated PKD phosphorylation, cultures were
pre-incubated with a calcium chelator (BAPTA-AM, 10 μM), an inhibitor of IP3R-mediated
calcium release (xestospongin C, 1 μM), or one of two PKC inhibitors (GF109203X and
Go6976, 10 μM each). Of these, the PKC inhibitor GF109203X most potently reduced
DHPG-induced PKD phosphorylation (Fig. 2d and Table 1;two-way ANOVA for inhibitor,
p<0.0001; inhibitor×stimulation interaction, p<0.0001), although as with the PLC inhibitor,
a small DHPG-induced increase was still detected (post-hoc analysis GF109203X/control
vs. GF109203X/DHPG, p<0.01). The PKC inhibitor Go6976 also significantly, but not
completely, reduced DHPG-induced PKD phosphorylation (Table 1; two-way ANOVA for
inhibitor, p<0.0001; inhibitor×stimulation interaction, p<0.0001; post-hoc analysis Go6976/
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control vs. Go6976/DHPG, p<0.0001). By contrast, neither the calcium chelator BAPTAAM nor the IP3R inhibitor xestospongin C decreased DHPG-induced PKD phosphorylation.
In fact, there was a slight increase in PKD phosphorylation in the presence of BAPTA-AM
(Table 1; two-way ANOVA for inhibitor, p<0.05; inhibitor×stimulation interaction,
p=0.203), whereas xestospongin C had no effect (Table 1; two-way ANOVA for inhibitor,
p=0.547; inhibitor×stimulation interaction, p=0.116). These data suggest that activation of
PKC, but not calcium release, is required for DHPG-induced PKD phosphorylation.
DHPG-Induced Phosphorylation of PKD Is Modulated by ERK1/2 and Possibly p38 MAPK
Pathways
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It is possible that the slight increase in PKD phosphorylation remaining in the presence of
PLC and PKC inhibitors may occur indirectly through other pathways activated by mGluR5
stimulation. A number of pathways have been reported to lie downstream of group I
mGluRs, including the MEK/ERK1/2 pathway, the p38 MAPK pathway, the PI3K/Akt/
mTOR pathway, and Src and other tyrosine kinases (Gerber et al. 2007). To test this
hypothesis, cultures were pre-incubated with inhibitors to each of these targets prior to
DHPG stimulation (Table 1). None of the inhibitors significantly reduced DHPG-induced
PKD phosphorylation. This suggests that the remaining PKD phosphorylation is not due to
activation of any of these individual pathways (although we cannot rule out that a
combination of pathways may be involved). Interestingly, however, the MEK inhibitor
U0126 (10 μM) caused a significant enhancement of DHPG-induced PKD phosphorylation,
implying that the MEK/ERK1/2 pathway modulates PKD phosphorylation through a
feedback mechanism following group I mGluR stimulation (two-way ANOVA for inhibitor,
p<0.05; inhibitor×stimulation interaction, p<0.05; post-hoc analysis control/DHPG vs.
U0126/DHPG, p<0.05). Similarly, a trend towards an increase was observed in the presence
of the p38 MAPK inhibitor SB203580 (10 μM, two-way ANOVA for inhibitor, p=0.297;
inhibitor×stimulation interaction, p=0.087; post-hoc analysis control/DHPG vs. SB203580/
DHPG, p< 0.05). In contrast, the PI3K inhibitor wortmannin (1 μM), the mTOR inhibitor
rapamycin (1 μM), the Src inhibitor PP2 (25 μM), and the general tyrosine kinase inhibitor
genistein (100 μM) had no significant effect.
DHPG-Induced PKD Phosphorylation Is not Dependent on Network Activity or Activation
of Ionotropic Glutamate Receptors
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One consequence of the activation of mGluR5 is an increase in neuronal excitability and
network activity (Netzeband et al. 1997; Ireland and Abraham 2002), and it is possible that
PKD phosphorylation is an indirect result of this increase in network activity in the
hippocampal culture, rather than being caused directly by PLCβ and PKC activation. To
investigate this possibility, cultures were pre-incubated with an inhibitor of the sodium
channels required for the generation of action potentials (TTX, 1 μM), an inhibitor of
AMPA/kainate-type ionotropic glutamate receptors (CNQX, 50 μM), or an inhibitor of the
NMDA-type ionotropic glutamate receptors (APV, 100 μM), all of which are known to
contribute to neuronal network activity. However, no significant effect on DHPG-induced
PKD phosphorylation was observed with any of these inhibitors (Table 1), suggesting that
PKD phosphorylation is likely to be a direct consequence of PLCβ and PKC activation,
rather than an indirect effect of increased network activity.
In Hippocampal Slices, mGluR-Induced PKD Phosphorylation May Occur Specifically at
Synapses
To determine whether DHPG-induced PKD phosphorylation is a phenomenon specific to
dissociated neurons, or whether it also occurs in more structurally intact preparations, PKD
phosphorylation was assessed in acute hippocampal slices. Slices were stimulated for 5 min
with 100 μM DHPG, and PKD phosphorylation levels were subsequently analyzed in whole
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tissue homogenate and in synaptoneurosomes, a preparation used to isolate the synaptic
component of the signal. Interestingly, unlike in dissociated cultures, no increase in PKD
phosphorylation was observed at the whole homogenate level (Fig. 3a; Student's t test, not
significant). However, PKD phosphorylation was significantly increased in
synaptoneurosomes following DHPG stimulation, albeit at a substantially lower level than
that observed in dissociated cultures (Fig. 3b; Student's t test, p<0.05). These data suggest
that group I mGluR stimulation of PKD phosphorylation is not unique to dissociated
neurons, but that in an intact network, this phosphorylation may occur specifically in the
synaptic compartment and may be restricted in magnitude by the local cellular environment.
Conclusions
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In this study, we report for the first time that stimulation of group I mGluRs in hippocampal
neurons results in phosphorylation of PKD at a site thought to correlate with PKD catalytic
activity. Together, our data provide an important basis for further investigation into the role
of PKD in mGluR5-mediated plasticity. Given the known cellular functions of PKD, there
are several potential mechanisms by which PKD may effect the molecular alterations
induced by mGluR5 activation. PKD is a regulator of polarized membrane trafficking, and
trafficking of glutamate receptors to and from the plasma membrane is a central element of
synaptic plasticity (Malenka and Bear 2004). In addition, PKD plays a role in regulating
transcription through its effect on histone deacetylases and stimulation of mGluRs results in
transcriptional activation (Gerber et al. 2007) that could potentially be enhanced by
concomitant PKD-mediated alterations in chromatin structure. Detailed studies will be
necessary to identify how these and other potential mechanisms may link mGluR5-induced
PKD phosphorylation to synaptic plasticity or related mGluR-mediated cellular functions.
Acknowledgments
We would like to thank Kathleen Oram, Zachary Cohen, Erik Sklar, and Suzanne Meagher for excellent technical
and administrative assistance. This work was supported by grants from HHMI, FRAXA, NIMH, NICHD, and the
Simons Foundation.
Abbreviations
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ACSF
Artificial cerebrospinal fluid
ANOVA
Analysis of variance
DAG
Diacylglycerol
DHPG
3,5-Dihydroxyphenylglycine
mGluR
Metabotropic glutamate receptor
PKC
Protein kinase C
PKD
Protein kinase D
PLC
Phospholipase C
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Krueger et al.
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Figure 1.
DHPG stimulation induces phosphorylation of PKD that is rapid and long lasting.
Hippocampal neurons were stimulated with DHPG for 5 min, followed by incubation with
DHPG-free medium for the indicated periods of time. a Immunoblot of phospho-PKD
(Ser-916) and total PKD at 5, 15, 30, and 60 min after onset of stimulation with 100 μM
DHPG. b Quantification of PKD phosphorylation at 5, 15, 30, and 60 min after onset of
stimulation with 100 μM DHPG (gray circles) or control (black circles)(n=8). c
Quantification of total PKD levels at 5, 15, 30, and 60 min after onset of stimulation with
DHPG or control. d Quantification of PKD phosphorylation at 0.5, 1, 2.5, and 5 min after
onset of stimulation with 100 μM DHPG (gray circles) or control (black circles)(n=8). e
Quantification of PKD phosphorylation at 1, 3, 6, and 24 h after onset of stimulation with
100 μM DHPG (gray circles) or control (black circles)(n=10). f Quantification of PKD
phosphorylation at 5 min after stimulation with control, 1, 10, 50, or 100 μM DHPG (n=7).
All data are expressed as percent control at each time point. The symbol asterisk indicates a
significant difference between control- and DHPG-treated samples. Error bars indicate
SEM
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Figure 2.
DHPG-induced PKD phosphorylation is dependent on activation of mGluR5, PLC, and
PKC, but not mGluR1. Hippocampal neurons were pre-incubated for 30 min with inhibitors
of a mGluR5 (MPEP, n=7), b mGluR1 (LY367385, n=7), c PLC (U73122, n=6), and d PKC
(GF109203X, n=7), followed by stimulation with 100 μM DHPG for 5 min. Black bars
represent control-treated samples, and gray bars represent DHPG-treated samples. All data
are expressed as percent control/control (exact values are reported in Table 1). The symbol
asterisk indicates a significant difference between control/DHPG- and inhibitor/DHPGtreated samples. The number sign indicates a significant difference between inhibitor/
control- and inhibitor/DHPG-treated samples. Error bars indicate SEM
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Figure 3.
DHPG-induced PKD phosphorylation in hippocampal slices is restricted to the synaptic
compartment. Hippocampal slices were stimulated with 100 μM DHPG for 5 min, then
analyzed for PKD phosphorylation in a whole tissue slices (n=6) and b synaptoneurosomes
(n=6). Black bars represent control-treated samples; gray bars represent DHPG-treated
samples. All data are expressed as percent control. The symbol asterisk indicates a
significant difference between control- and DHPG-treated samples. Error bars indicate
SEM
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PKC
PKC
Calcium
IP3R
MEK1/2
p38 MARK
PI3K
mTOR
Src
Tyr kinases
Na+
AMPAR
NMDAR
GF109203X
Go6976
BAPTA-AM
Xestospogin C
U0126
SB203580
Wortmannin
Rapamycin
PP2
Genistein
TTX
CNQX
APV
100
50
1
100
25
1
1
10
10
1
10
10
10
50
5
5
5
5
5
5
7
7
7
5
5
7
7
6
7
100±24.6
100±24.6
100±24.6
100±12.6
100±12.6
100±16.5
100±12.9
100±12.9
100±12.9
100±33.8
100±34.1
100±12.0
100±10.4
100±15.0
100±4.8
100±5.3
1297.2±67.0
1297.2±67.0
1297.2±67.0
623.8±43.4
623.8±43.4
992.9±156.1
888.5±26.6
888.5±26.6
888.5±26.6
1291.4±99.6
1286.2±72.1
804.7±26.1
825.1±61.5
783.8±34.2
1198.6±43.4
1193.2±29.7
116.2±9.0
106.9±25.8
151.9±18.9
83.0±30.4
83.2±20.4
92.7±14.0
73.1±23.6
79.1±15.7
116.3±16.9
168.0±40.9
164.0±48.0
86.3±9.6
70.6±8.5
93.8±19.0
80.2±16.9
77.0±12.8
Inhibitor/
Control
1369.6±37.4
1341.9±137.4
1156.2±101.5
695.9±62.5
636.9±36.8
918.3±73.5
828.6±44.0
978.6±26.3
1121.2±93.1
1143.3±64.6
1539.9±108.4
552.5±21.9
291.5±61.3
291.0±12.9
1226.3±44.3
120.3±22.1
Inhibitor/
DHPG
1
1
1
1
1
1
1
1
1, 2, 3
1
1, 2
1, 2, 3
1, 2, 3
1, 2, 3
1
1, 2, 3
ANOVA
Statistics
2
2
2
2
2
2
2
1, 2
1, 2
2
2
1, 2
1, 2
1, 2
2
1
Post-hoc
Hippocampal neurons were pre-incubated for 30 min with inhibitors as indicated, followed by stimulation with 100 μM DHPG for 5 min. All data are expressed as percent control/control. For the ANOVA
statistics, “1” indicates a significant main effect of stimulation, “2” indicates a significant main effect of inhibitor, and “3” indicates a significant stimulation×inhibitor interaction. For the post-hoc tests, “1”
indicates a significant difference between control/DHPG- and inhibitor/DHPG-treated samples, and “2” indicates a significant difference between inhibitor/control- and inhibitor/DHPG-treated samples
channel
PLC
U73122
50
7
mGluR1
LY367385
2
mGluR5
MPEP
Control/
DHPG
Control/
control
n
Target
Name
Concentration (μM)
Phospho-PKD levels
Inhibitor
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Summary data for inhibitor experiments
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Table 1
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